Detection of point mutation, so-called single nucleotide polymorphism (SNP), is employed widely as a method of analyzing, at the gene level, for example, the causes of all types of diseases and the individual differences in disease liability (susceptibility to diseases) and in drug action.
Examples of the common methods of detecting a point mutation include: (1) a direct sequencing method in which the region corresponding to a sequence to be detected in the target DNA of a sample is amplified and all the gene sequences are analyzed, (2) a pyrosequencing method, (3) a denaturing HPLC method in which the region corresponding to a sequence to be detected is amplified, HPLC is performed in a temperature gradient column, and the presence or absence of any mutation is detected according to the time that is required for elution, (4) an invader method in which, using fluorescence that is emitted when a fluorescent probe binds to the region containing a target mutation, a mutation is detected through detection of the fluorescence, and (5) the ASP-PCR method in which PCR is performed using a primer with a target mutation located at the 3′-end region and the mutation is judged depending on the presence or absence of amplification.
However, the aforementioned methods (1), (2), and (4) are not very sensitive, specifically their sensitivity is approximately 20%, approximately 5%, and approximately 5%, respectively, and they require a considerable amount of time and labor for their operations. Furthermore, in the aforementioned method (3), the sensitivity is as low as approximately 10%, it only can check the presence or absence of a mutation, and it cannot analyze the site and type of a mutation. Therefore, there is a problem in that it lacks specificity. The aforementioned method (5) is highly sensitive but is less specific, so that it is apt to give a false-positive result, which is a problem. In this context, the lower the numerical value (%) is, the relatively higher the sensitivity.
Because of these problems, recently, a method of analyzing the melting temperature (Tm) of double-stranded nucleic acid formed of a probe and target nucleic acid is used as a method of detecting a point mutation. Since this method is performed through, for example, Tm analysis or analysis of the melting curve of the double strand, it is referred to as melting curve analysis. This is a method as described below. That is, first, a probe complementary to a sequence to be detected containing a target point mutation is used to form a hybrid (double-stranded DNA) between the aforementioned probe and a target single-stranded DNA in a detection sample. Subsequently, this hybridization product is heat-treated, so that dissociation (melting) of the hybrid accompanying the temperature rise is detected by a change in the signal such as absorbance. The Tm value is then determined based on the result of the detection and the presence or absence of any point mutation is judged accordingly. The higher the Tm value, the higher the homology of the hybridization product, and the lower the Tm value, the lower the homology. Therefore the Tm value (reference value for assessment) is determined beforehand for the hybridization product between the sequence to be detected containing a point mutation and a probe complementary thereto and then the Tm value (measured value) of the hybridization product between the target single-stranded DNA in the detection sample and the aforementioned probe is measured. When the measured value is equal to the reference value, it is considered as matching, that is, it can be judged that the point mutation is present in the target DNA. On the other hand, when the measured value is lower than the reference value, it is considered as mismatching, that is, it can be judged that no point mutation is present in the target DNA.
However, such a detection method using Tm analysis also has a problem in that the sensitivity is low. This is a problem, particularly, in detecting point mutations in DNAs derived from blood cells of leukemia patients (Patent Document 1). Leukemia is a disease resulting from malignant transformation of hematopoietic stem cells in the bone marrow. Chronic myeloid leukemia (CML), among others, is known to have its origin in the bcr-abl fusion gene generated by translocation between chromosome 9 and chromosome 22 and, for example, imatinib, an ABL kinase inhibitor, is used widely for the treatment of the disease. However, when a point mutation is present in the abl gene (including the abl gene in the aforementioned fusion gene), resistance to imatinib is developed, which is a problem. In that case, an increase in dose of imatinib, a change to some other therapeutic drug, or switching to, for example, bone marrow transplantation becomes necessary for the treatment. Therefore in the treatment of leukemia, particularly CML, it is very important to detect the presence or absence of a point mutation in the abl gene. However, even in the blood of the same CML patient, there are blood cells with point mutations that have occurred in the abl genes (mutated genes) and those with no point mutations that have occurred in the abl genes (normal genes), and the difference between them resides merely in a point mutation, that is, a single base of the sequence. Then a phenomenon may occur where the probe for detecting a point mutation hybridizes (perfectly matches) with the mutated sequence (the sequence to be detected) containing the point mutation and also hybridizes (mismatches) with a normal sequence (sequence not to be detected) that does not contain the point mutation. In such a case, when a melting curve that indicates the relationship between signal intensity and temperature is prepared based on Tm analysis, it is difficult to detect the peak on the higher temperature side that corresponds to the perfectly matched mutated sequence due to the presence of the peak on the lower temperature side that corresponds to the mismatched normal sequence, which is a problem. That is, even when a mutated sequence containing a mutation is present, the presence of a normal sequence containing no mutation makes it difficult for the conventional probe to detect the presence of the mutated sequence, which causes a decrease in detection sensitivity.
[Patent Document 1] JP 2004-537992 A